Mitosis must be carried out with high fidelity to ensure that each daughter cell receives a complete complement of the genome. Bipolar spindle assembly is directed by the duplication of the centrosome, which serves as the major microtubule-organizing center in mammalian cells. At the core of each centrosome is a pair of centrioles, composed of triplet microtubules: the duplication and assembly of the centrioles mirrors the duplication of the centrosome as a whole. Mistakes in the duplication of the centrosome itself can lead to the formation of multipolar spindles that do contribute to genomic instability. Indeed, many human tumor cells have abnormal numbers of centrosomes. To continue the investigation of the cell cycle regulation of centrosome duplication we will use live-cell video microscopy and biophysical/cell biological techniques to analyze centrosome reproduction in mammalian cells and frog egg extracts. These studies are intended to reveal important novel aspects of centrosome function that have proved difficult to study using molecular genetic or biochemical approaches. In Aim I, we will determine if Cdk2-cyclin E licenses centrosome duplication in Xenopus egg extracts, as suggested by our previous work. We will also test whether centrosome re-duplication in cells arrested in S-phase requires licensing. Finally, we test the hypothesis that centrosome duplication does not require the onset of S-phase, also suggested by our previous studies. In Aim II, we will investigate centriole assembly. Centriole microtubules are thought to be very stable, but our preliminary work on the centriole protein tektin suggests they undergo turnover throughout the cell cycle. We will use FRAP to measure the rate of GFPtektin/ centriole microtubule turnover in living cells. We will also test the hypothesis that tektin, a core centriole component, is synthesized coordinate with centrosome duplication, suggesting a mechanism for the cell cycle regulation of duplication itself. We will also test whether knocking-down the expression of tektin leads to inhibition of centriole duplication/elongation, and whether this induces cell cycle arrest. In Aim III, we examine early events in centrosome reproduction. We have developed methods to assemble centrosome precursors in mammalian cells. We will examine the kinetics of precursor formation using live-cell microscopy. We will isolate these precursor structures, analyze their protein composition by multi-LC Mass Spectrometry, and clone candidate genes. Using these, we will test whether the initial duplication event (precursor formation) is coordinated with centriole assembly or occurs earlier in the cell cycle. A detailed understanding of the regulation of centrosome reproduction will advance our knowledge of cancer biology - a disease characterized by unregulated cell proliferation. Our experiments should complement the genetic and biochemical work currently being done in a variety of model systems, and ultimately provide a fertile source of potential targets for the next generation of anti-cancer drugs.